Methods and compositions for targeted single-stranded cleavage and targeted integration

ABSTRACT

Disclosed herein are methods and compositions for generating a single-stranded break in a target sequence, which facilitates targeted integration of one or more exogenous sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 16/875,152, filed May 15, 2020, which is acontinuation of U.S. patent application Ser. No. 16/146,637, filed Sep.28, 2018, now U.S. Pat. No. 10,689,717, which is a continuationapplication of U.S. patent application Ser. No. 15/495,325, filed Apr.24, 2017, now U.S. Pat. No. 10,113,207, which is a continuationapplication of U.S. patent application Ser. No. 14/925,386, filed Oct.28, 2015, now U.S. Pat. No. 9,631,186, which is a continuationapplication of U.S. patent application Ser. No. 14/199,753, filed Mar.3, 2014, now U.S. Pat. No. 9,200,266, which is a continuationapplication of Ser. No. 12/583,329, filed Aug. 18, 2009, now U.S. Pat.No. 8,703,489, which claims the benefit of U.S. Provisional ApplicationNo. 61/189,800, filed Aug. 22, 2008, which are all incorporated byreference in their entireties.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the fields of genome engineering, genetargeting, targeted chromosomal integration and protein expression.

BACKGROUND

A major area of interest in genome biology, especially in light of thedetermination of the complete nucleotide sequences of a number ofgenomes, is the targeted alteration of genome sequences.

Artificial nucleases, which link the cleavage domain of nuclease to adesigned DNA-binding protein (e.g., zinc-finger protein (ZFP) linked toa nuclease cleavage domain such as from FokI), have been used fortargeted cleavage in eukaryotic cells. For example, zinc fingernuclease-mediated genome editing has been shown to modify the sequenceof the human genome at a specific location by (1) creation of adouble-strand break (DSB) in the genome of a living cell specifically atthe target site for the desired modification, and by (2) allowing thenatural mechanisms of DNA repair to “heal” this break.

To increase specificity, the cleavage event is induced using one or morepairs of custom-designed zinc finger nucleases that dimerize uponbinding DNA to form a catalytically active nuclease complex. Inaddition, specificity has been further increased by using one or morepairs of zinc finger nucleases (ZFNs) that include engineered cleavagehalf-domains that cleave double-stranded DNA only upon formation of aheterodimer. See, e.g., U.S. Patent Publication No. 2008/0131962,incorporated by reference herein in its entirety.

The double-stranded breaks (DSBs) created by artificial nucleases havebeen used, for example, to induce targeted mutagenesis, induce targeteddeletions of cellular DNA sequences, and facilitate targetedrecombination at a predetermined chromosomal locus. See, for example,U.S. Patent Publication Nos. 2003/0232410; 2005/0208489; 2005/0026157;2005/0064474; 2006/0188987; 2006/0063231; 2007/0218528; 2007/0134796;and 2008/0015164 and International Patent Publication Nos. WO 07/014275and WO 2007/139982, the disclosures of which are incorporated byreference in their entireties for all purposes. Thus, the ability togenerate a DSB at a target genomic location allows for genomic editingof any genome.

There are two major and distinct pathways to repair DSBs—homologousrecombination and non-homologous end-joining (NHEJ). Homologousrecombination requires the presence of a homologous sequence as atemplate (e.g., “donor”) to guide the cellular repair process and theresults of the repair are error-free and predictable. In the absence ofa template (or “donor”) sequence for homologous recombination, the celltypically attempts to repair the DSB via the unpredictable anderror-prone process of non-homologous end-joining (NHEJ).

Single strand breaks (SSBs), including DNA nicks, are one of the mostfrequent DNA lesions produced by endogenous reactive oxygen species andduring DNA metabolism, such as DNA repair and replication. See,McKinnon, et al. (2007) Annu Rev Genomics Hum Genet 8:37-55; Okano, etal. (2003) Mol Cell Biol 23:3974-3981. Chromosomes of non-apoptoticcells contain single-strand discontinuities (SSBs/nicks) positioned atabout 50 kb intervals all over the entire genome. See, e.g., Szekvolgyi,et al. (2007) Proc Natl Acad Sci USA 104:14964-14969. Most SSB/nicks arerepaired by a rapid global SSB repair process that can be divided intofour basic steps: SSB detection by poly (ADP-ribose) polymerase-1(PARP-1), DNA end processing by various enzymes, DNA gap filling by DNApolymerases, and DNA ligation by DNA ligases, See Caldecott, K. W.(2008) Nat Rev Genet 9:619-631. Lee, et al. (2004) Cell 117:171-184found data to suggest that nicks induced by mutated RAG proteins mightinitiate homology-directed repair (HDR) in mammalian cells.

However, it has not previously been shown that ZFNs can be engineered toinduce SSBs/nicks, or that these SSBs/nicks can be repaired byhomologous recombination, or that they can be used to facilitate thetargeted integration of a transgene via homologous recombination. Thus,there remains a need for methods and composition that generatesingle-stranded breaks (nicks) in double-stranded DNA and facilitatetargeted integration by homologous recombination at the nicked site,without simultaneously occurrence of error-prone NHEJ repair inmammalian/human cells.

SUMMARY

Disclosed herein are methods and compositions for inducing a targetedsingle-stranded break in any double-stranded target sequence ofinterest. Also described are methods of facilitating homologousrecombination and targeted integration following single-strandedcleavage of a target. Thus, targeted modulation of a genome isdescribed.

In one aspect, artificial (non-naturally occurring) nucleases thatgenerate single-stranded cuts in a desired double-stranded targetsequence are provided. The nucleases described herein comprise aDNA-binding domain (e.g., engineered zinc finger protein) and at leastone cleavage domain or at least one cleavage half-domain. In certainembodiments, the nucleases are zinc finger nucleases comprising a zincfinger domain that is engineered to bind any selected sequence (e.g.,gene). Any of the zinc finger proteins described herein may include 1,2, 3, 4, 5, 6 or more zinc fingers, each zinc finger having arecognition helix that binds to a target subsite in the selectedsequence(s) (e.g., gene(s)). The cleavage domain can be derived from anynuclease, for example, a cleavage half-domain from a Type IIS nuclease,such as FokI. In certain embodiments, the cleavage half-domain comprisesan engineered FokI cleavage-half domain that forms a heterodimer withanother cleavage half-domain (e.g., engineered or wild-type in thedimerization domain). In still further embodiments, the cleavage domain(e.g., engineered FokI cleavage half-domain) comprises a mutation thatinactivates the catalytic domain (enzymatic activity) of the nucleasedomain.

In another aspect, provided herein are complexes and/or compositionscomprising a pair of zinc finger nucleases, each nuclease comprising anengineered zinc finger domain and a FokI cleavage half-domain, whereinthe cleavage half-domain forms a dimer (homodimer or heterodimer). Incertain embodiments, one zinc finger nuclease of the pair comprises afirst catalytically active engineered cleavage-half domain and the otherzinc finger nuclease comprises a second catalytically inactiveengineered cleavage half-domain, in which the first and secondengineered cleavage half-domains form an obligate heterodimer.

In yet another aspect, a polynucleotide encoding any of the nucleasesdescribed herein is provided.

In yet another aspect also provided is an isolated cell comprising anyof the proteins and/or polynucleotides described herein. In certainembodiments, cell lines in which exogenous sequences have beenintroduced via targeted alteration are provided. In certain embodiments,one or more selected gene(s) are inactivated (partially or completely)in these cell lines. In other embodiments, the cells or cell linesinclude one or more transcribed and/or translated exogenous sequencesthat have been stably or transiently introduced into the cells. Suchcell lines are generated by culturing of cells comprising any of theproteins and/or polynucleotides described herein in a cell lineresulting in the inactivation of selected gene(s).

Also provided are transgenic organisms comprising one or moretranscribed and/or translated exogenous sequences that have been stablyor transiently introduced into the cells. Further provided aretransgenic organisms that contain gene(s) selectively inactivated by themethods provided herein, or organisms containing exogenous sequencescapable of altering the expression of endogenous sequences. Transgenicorganisms as described herein may be plants (e.g. crop plants or tobaccostrains) or animals (e.g. mice, rats, rabbits, fish, etc.). In certainembodiments, the transgenic organisms are used to generate lines oforganisms carrying one or more sequences encoding nucleases as describedherein and/or one or more exogenous sequences (e.g., sequences insertedinto the genome via targeted integration using nucleases as describedherein). For instance, disclosed herein are transgenic plants and plantlines comprising nucleases as described herein under the control of aninducible promoter. Accordingly, transgenic plants and plant linescomprising episomal or integrated sequences encoding nucleases can beexpressed in the plant at the desired time and/or in the desired tissueof the plant.

In addition, methods of generating a specific single-stranded break in atarget double-stranded sequence in a cell are provided. In certainembodiments, the methods involve introducing one or more pairs ofnucleases (proteins or polynucleotide(s) encoding the nucleases) intothe cell. Each nuclease pair comprises a first nuclease with a firstDNA-binding domain and a first catalytically active cleavage-half domainand a second nuclease with a second DNA-binding domain and secondcatalytically inactive cleavage-half domain. The one or more pairs ofnucleases are introduced into the cells under conditions such that thefirst and second cleavage half-domains of each pair form a dimer andgenerate a single-stranded break in the target sequence.

In certain embodiments, the first and/or second DNA-binding domainscomprise a zinc finger protein (e.g., an engineered zinc fingerprotein). Furthermore, in any of the methods described herein, the firstand second cleavage half-domains can comprise FokI cleavagehalf-domains, for example FokI cleavage half-domains that are engineeredto form obligate heterodimers. The target sequence can be anydouble-stranded sequence, for example, a sequence in cellular chromatinsuch as a genomic sequence or portion thereof. In addition, the targetsequence can be an extrachromosomal double stranded DNA sequence, forexample in a plasmid or virus. Similarly, the target sequence can be inany cell type including, prokaryotic and eukaryotic cells, such asfungal cells, plant cells, animal cells, mammalian cells, primate cellsand human cells.

The site of single-stranded break can be coincident with the sequence towhich the catalytically active nuclease binds, or it can be adjacent(e.g., separated from the near edge of the binding site by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides). The fusionproteins can be expressed in a cell, e.g., by delivering the fusionproteins to the cell and/or by delivering a polynucleotide encoding oneor more nucleases to a cell, wherein the polynucleotide, if DNA, istranscribed into mRNA which is then translated into the fusion protein.Alternatively, an RNA molecule can be delivered to the cell which thenis translated to generate the fusion protein. Methods for polynucleotideand polypeptide delivery to cells are presented elsewhere in thisdisclosure.

Methods for targeted recombination (for, e.g., alteration or replacementof a sequence in a chromosome or a region of interest in cellularchromatin) are also provided. For example, a mutant genomic sequence canbe replaced by a wild-type sequence, e.g., for treatment of geneticdisease or inherited disorders. In addition, a wild-type genomicsequence can be replaced by a mutant sequence, e.g., to prevent functionof an oncogene product or a product of a gene involved in aninappropriate inflammatory response. Furthermore, one or more alleles ofa gene can be replaced by one or more different alleles (e.g., biallelictargeted integration).

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a single-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thesingle-stranded break has been shown herein to facilitate integration ofthe donor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In addition, a method for replacement of a region of interest incellular chromatin (e.g., a genomic sequence) with a first nucleotidesequence is provided, the method comprising: (a) engineering a firstzinc finger binding domain to bind to a second sequence in the region ofinterest; (b) providing a second zinc finger binding domain to bind to athird sequence in the region of interest; (c) expressing a first fusionprotein in a cell, the first fusion protein comprising the first zincfinger binding domain and a first catalytically active cleavagehalf-domain; (d) expressing a second fusion protein in the cell, thesecond fusion protein comprising the second zinc finger binding domainand a second catalytically inactive cleavage half-domain; and (e)contacting the cell with a polynucleotide comprising the firstnucleotide sequence; wherein the first fusion protein binds to thesecond sequence and the second fusion protein binds to the thirdsequence, thereby positioning the cleavage half-domains such that asingle-stranded break is made in cellular chromatin in the region ofinterest and a nucleotide sequence in the region of interest is replacedwith the first nucleotide sequence. In certain embodiments, thesingle-stranded break in cellular chromatin is made in the region ofinterest at a site between the second and third sequences. The zincfinger nucleases may be provided to the cells as proteins and/or as oneor more polynucleotides encoding said zinc finger nuclease(s). Forexample, two polynucleotides, each comprising sequences encoding one ofthe two fusion proteins, can be introduced into a cell. Alternatively, asingle polynucleotide comprising sequences encoding both fusion proteinscan be introduced into the cell.

In any of the methods described herein, additional pairs of zinc fingerproteins can be used for additional double-stranded and/orsingle-stranded cleavage of additional target sites within the cell.

Thus, in one embodiment, a method for replacement of a region ofinterest in cellular chromatin (e.g., a genomic sequence) with a firstnucleotide sequence comprises: (a) engineering a first zinc fingerbinding domain to bind to a second sequence in the region of interest;(b) providing a second zinc finger binding domain to bind to a thirdsequence; and (c) contacting a cell with: (i) a first polynucleotidecomprising the first nucleotide sequence; (ii) a second polynucleotideencoding a first fusion protein, the first fusion protein comprising thefirst zinc finger binding domain and a first catalytically activecleavage half-domain; and (iii) a third polynucleotide encoding a secondfusion protein, the second fusion protein comprising the second zincfinger binding domain and a second catalytically inactive cleavagehalf-domain; wherein the first and second fusion proteins are expressed,the first fusion protein binds to the second sequence and the secondfusion protein binds to the third sequence, thereby positioning thecleavage half-domains such that a single-stranded break is generated incellular chromatin in the region of interest; and the region of interestis replaced with the first nucleotide sequence.

In the preferred embodiments of methods for targeted recombinationand/or replacement and/or alteration of a sequence in a region ofinterest in cellular chromatin, a chromosomal sequence is altered byhomologous recombination with an exogenous “donor” nucleotide sequence.Such homologous recombination is stimulated by the presence of asingle-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present. Notably, single-strand breaks incellular chromatin do not stimulate cellular mechanisms ofnon-homologous end joining.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, for example sequences such asthose found in artificial chromosomes, that are homologous or identicalto sequences in the region of interest. In other embodiments, the donorsequence is non-homologous to the first sequence, and is inserted intothe genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided. Further still, the plant cell lines with partially orcompletely inactivated genes can be used to generate transgenic plants.Plant cell lines comprising nucleases as described herein, whereinexpression of the nuclease is driven by an inducible promoter are alsoprovided. Similarly, mammalian germ cells, such as oocytes, withpartially or completely inactivated genes can be used to generatetransgenic animals.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or noncoding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

In any of the cells, cell lines and methods described herein, the cellor cell line can be a COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11, CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK,HaK, NSO, SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T),PerC.6® (Crucell); EBx™ (Sigma-Aldrich Group), insect cells such asSpodoptera fugiperda (Sf), or fungal cells such as Saccharomyces, Pichiaand Schizosaccharomyces.

In another aspect, the invention provides kits comprising one or morenucleases (or polynucleotides encoding these nucleases) as describedherein for carrying out the methods as described herein. The kits mayoptionally comprise reagents, buffers, cells, suitable containers andwritten instructions.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting an exemplary zinc finger nuclease (ZFN)architecture that includes an inactivated cleavage domain. The ZFN paircleaves only one DNA strand to form single-stranded breaks (SSBs), alsoreferred to as nicks.

FIGS. 2A through 2C depict single-stranded cleavage of a double-strandedtarget using a pair of zinc finger nucleases where one nuclease includesa cleavage domain that has been mutated to inactivate cleavage activityof the nuclease domain.

FIG. 2A depicts analysis of double-stranded cleavage products obtainedby cleavage of a target substrate with the indicated ZFN pairs. Fromleft to right, lanes show: molecular weight ladder, control, aCCR5-targeted ZFN pair designated 8196zKK:8267EL, where KK and EL referto engineered cleavage domains that form obligate heterodimers togenerate double-stranded breaks (see, U.S. Patent Publication2008/0131962), referred to as wildtype 8196zKK8267EL (WT) in thisapplication; a CCR5-targeted ZFN pair designated 8196zKK:8267EL, wherethe 8267EL protein also contains a mutation at position 450 of thecatalytic domain (D450N) of one cleavage half-domain that inactivatesone cleavage half-domain of the ZFN pair; a CCR5-targeted ZFN pairdesignated 8196zKK:8267EL, where the 8267EL protein also contains amutation at position 467 in the catalytic domain (D467A) thatinactivates one cleavage half-domain of the ZFN pair; a second molecularweight ladder, a second control; a CCR5-targeted ZFN pair designated8267RD:8196zDR, where RD and DR refer to engineered cleavage domainsthat form obligate heterodimers (see, U.S. Patent Publication No.2008/0131962), referred to as wildtype 8267RD:8196zDR (WT) in thisapplication; a CCR5-targeted ZFN pair designated 8267RD:8196zDR, wherethe 8196zDR protein also contains a mutation at position 450 of thecatalytic domain (D450N) of one cleavage half-domain that inactivatesone cleavage half-domain of the ZFN pair; a CCR5-targeted ZFN pairdesignated 8267RD:8196zDR, where the 8196zDR protein also contains amutation at position 467 in the catalytic domain (D467A) thatinactivates one cleavage half-domain of the ZFN pair.

FIG. 2B depicts analysis of single-stranded cleavage products obtainedby cleavage of a target substrate with the indicated ZFN pairs. Asdescribed above for FIG. 2A, from left to right, lanes show: molecularweight ladder, control, a CCR5-targeted ZFN pair designated8196zKK:8267EL(WT); the CCR5-targeted ZFN pair designated 8196zKK:8267EL(D450N); the CCR5-targeted ZFN pair designated 8196zKK:8267EL(D467A); asecond molecular weight ladder, a second control; the CCR5-targeted ZFNpair designated 8267RD:8196zDR (WT); the CCR5-targeted ZFN pairdesignated 8267RD:8196zDR (D450N); and the CCR5-targeted ZFN pairdesignated 8267RD:8196zDR (D467A).

FIG. 2C is a schematic depicting possible cleavage patterns using mutantcleavage domains as described herein, showing which bands will bedetected in the autoradiographical analysis due to the position of theradiolabel on the various DNA segments.

FIGS. 3A through 3E depict repair of ZFN-induced SSB/nicks in K562 cellsby an HDR-dependent single-strand annealing (SSA) pathway. K562 cellswere either untreated (Untreated, FIG. 3A) or transfected with a SSA-GFPreporter DNA plasmid in the absence (No ZFN, FIG. 3B) or presence of ZFNexpression plasmids as described above in FIG. 2A: the CCR5-targeted ZFNpair designated 8196zKK:8267EL (WT, FIG. 3C); the CCR5-targeted ZFN pairdesignated 8196zKK:8267EL (D450N, FIG. 3D); the CCR5-targeted ZFN pairdesignated 8196zKK:8267EL (D467A, FIG. 3E). Cells were collected 3 dayspost-transfection and subjected to flow cytometric analysis after 5 minincubation with propidium iodide (PI) to stain non-viable cells (Pr).Integration of the GFP donor sequence into the break in the CCR5 geneoccurs through an HDR-dependent pathway since the GFP donor sequence isflanked by CCR5 sequences that are homologous to regions flanking thenick site. Thus, an increase in the observed GFP fluorescent signalindicated targeted integration of the donor sequence has occurred.Percentage of cells in each quadrant is indicated at the upper corner ofquadrant. The data demonstrate that the ZNF pairs containing the D450Nand the D467A mutants are able to integrate the GFP sequences.

FIGS. 4A through 4D depict analysis of non-homologous end joining (NHEJ)and targeted integration (TI) of an exogenous sequence (patch donor)following the induction of double-stranded or single-stranded breaks inthe target gene. FIG. 4A depicts repair of double-stranded, but notsingle-stranded, breaks by NHEJ in K562 cells comprising the ZFN pairsindicated above each lane. The lane number corresponds to sample numberin Table 1. FIG. 4B depicts targeted integration of a 46 bp CCR-5 patchdonor molecule following single- or double-stranded cleavage of theCCR-5 gene with the ZFN pairs indicated in Table 1. Lane line numberscorrespond to sample numbers. FIGS. 4C and 4D depict homologousrecombination events in cells treated with a single zinc finger nucleaseor combinations of two zinc finger nucleases (indicated above eachlane).

FIGS. 5A and 5B depict analysis of targeted integration and NHEJ in K562cells transfected with the indicated ZFN combinations in the presence ofa 46 bp CCR5-patch donor. FIG. 5A shows targeted integration analysisand FIG. 5B shows NHEJ analysis. Numbers at the bottom of each laneindicate frequency (%) of targeted integration or NHEJ.

FIGS. 6A and 6B depict analysis of targeted integration and NHEJ in K562cells transfected with indicated ZFN combinations in the presence of aCCR5-tNGFR-outGFP donor. FIG. 6A shows NHEJ analysis and FIG. 6B showstargeted integration analysis by Southern blot. Numbers at the bottom ofeach lane indicate frequency (%) of targeted integration or NHEJ.

FIG. 7 is a graph depicting 53BP1+ foci (indicative of DSBs) at theindicated time points in K562 cells transfected with the indicated ZFNconstructs. Open circles depict control cells having no ZFNs; shadedcircles show cells transfected with wild-type ZFNs; open squares showcells transfected with one wild-type ZFN and one inactivated ZFN(D450N); and shaded squares show cells transfected with one wild-typeZFN and one inactivated ZFN (D467A).

FIG. 8 depicts analysis of targeted integration at the CXCR4 locus inK562 cells. K562 cells were nucleofected with a CXCR4-patch donor DNA inthe absence (no ZFN) or presence of CXCR4 D450N ZFNs:CXCR4-ZFN-L-EL-D450N+CXCR4-ZFN-R-KK. Cells were allowed to recover for4-7 days and then nucleofected again with the same DNAs. The process wasrepeated for a total of 4 nucleofections. Cells were then collected 3days after the last nucleofection for gDNA preparation and RFLP assay.Expected positions of CXCR4 modified by TI were indicated by arrows.Numbers at the bottom of each lane indicate frequency (%) of TI.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods useful for targetedsingle-stranded cleavage of cellular chromatin and for targetedalteration of a cellular nucleotide sequence, e.g., by targetedsingle-stranded cleavage followed by homologous recombination between anexogenous polynucleotide (comprising one or more regions of homologywith the cellular nucleotide sequence) and a genomic sequence. Genomicsequences include those present in chromosomes, episomes, organellargenomes (e.g., mitochondria, chloroplasts), artificial chromosomes andany other type of nucleic acid present in a cell such as, for example,amplified sequences, double minute chromosomes and the genomes ofendogenous or infecting bacteria and viruses. Genomic sequences can benormal (i.e., wild-type) or mutant; mutant sequences can comprise, forexample, insertions, deletions, translocations, rearrangements, and/orpoint mutations. A genomic sequence can also comprise one of a number ofdifferent alleles. The compositions and methods can also be used fortargeted alteration of extrachromosomal nucleotide sequences, e.g.plasmids.

Compositions useful for targeted single-stranded cleavage andrecombination include fusion proteins comprising a cleavage half-domainand a zinc finger binding domain, polynucleotides encoding theseproteins and combinations of polypeptides and polypeptide-encodingpolynucleotides. A zinc finger binding domain can comprise one or morezinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers), andcan be engineered to bind to any genomic or episomal sequence. Thus, byidentifying a target genomic or episomal region of interest at whichcleavage or recombination is desired, one can, according to the methodsdisclosed herein, construct one or more fusion proteins comprising botha catalytically active and catalytically inactive cleavage half-domainand a zinc finger domain engineered to recognize a target sequence insaid genomic or episomal region. The presence of such a fusion protein(or proteins) in a cell will result in binding of the fusion protein(s)to its (their) binding site(s) and single-stranded cleavage within ornear said genomic or episomal region. Notably, as shown herein, if anexogenous polynucleotide having regions homologous to the genomic orepisomal region is also present in such a cell, homologous recombinationoccurs at a high rate between the genomic or episomal region and theexogenous polynucleotide.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL,Second edition, Cold Spring Harbor Laboratory Press, 1989 and Thirdedition, 2001; Ausubel, et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons, New York, 1987 and periodic updates; the seriesMETHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS IN ENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger protein. Therefore, engineered zinc fingerproteins are proteins that are non-naturally occurring. Non-limitingexamples of methods for engineering zinc finger proteins are design andselection. A designed zinc finger protein is a protein not occurring innature whose design/composition results principally from rationalcriteria. Rational criteria for design include application ofsubstitution rules and computerized algorithms for processinginformation in a database storing information of existing ZFP designsand binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242;and 6,534,261; see also International Patent Publication Nos. WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; InternationalPatent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO 02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength. Alternately, a donor sequence can be an artificial chromosomesequence such as a bacterial artificial chromosome (BAC) or yeastartificial chromosome (YAC).

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides, an artificial chromosome, or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. With respect tosequences described herein, the range of desired degrees of sequenceidentity is approximately 80% to 100% and any integer valuetherebetween. Typically the percent identities between sequences are atleast 70-75%, preferably 80-82%, more preferably 85-90%, even morepreferably 92%, still more preferably 95%, and most preferably 98%sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acids, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

Conditions for hybridization are well-known to those of skill in the art(see, for example, Nucleic Acid Hybridization: A Practical Approach,editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.;IRL Press). Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Single-stranded cleavage refers to cleavage of one stand ofdouble-stranded DNA/RNA and double-stranded cleavage refers to cleavageof both strands (e.g., via two distinct single-stranded cleavageevents). In certain embodiments, fusion polypeptides are used fortargeted single-stranded DNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The terms “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; and2008/0131962 and U.S. Pat. No. 7,914,796, incorporated herein byreference in their entireties.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome” is a chromatin complex comprising all or a portion of thegenome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogeneous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequenced may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and one or more cleavage domains) and fusion nucleicacids (for example, a nucleic acid encoding the fusion protein describedsupra). Examples of the second type of fusion molecule include, but arenot limited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Gene inactivation refers to anyreduction in gene expression as compared to a cell that does not includea ZFP as described herein. Thus, gene inactivation may be complete(knock-out) or partial (e.g., a hypomorph in which a gene exhibits lessthan normal expression levels or a product of a mutant gene that showspartial reduction in the activity it influences).

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

A “cell line” refers to a population of cells established in tissueculture from a primary culture. Thus, cell lines generated using zincfinger nuclease(s) arise from a cell (or cell line) in which one or moretarget genes have been partially or completely inactivated by one ormore zinc finger nucleases and in which the progeny of the cell (or cellline) retain the partial or complete inactivation phenotype aftermultiple passages in culture. Furthermore, a cell or cell line is“deficient” in expression of one or more indicated genes when expressionof the gene(s) is(are) reduced (knock-downs) or eliminated (knockouts).

By “transgenic” is meant any animal or plant which includes a nucleicacid sequence which is introduced into a cell and becomes part of thegenome of the animal or plant. The term refers to a geneticallyengineered animal or plant as well as offspring of geneticallyengineered animals. The non-human transgenic animal includes vertebratessuch as rodents, non-human primates, sheep, dogs, cows, amphibians,birds, fish, insects, reptiles, etc. The term also includes animals andplants in which the introduced nucleic acid sequence is found, or inwhich the nucleic acid sequence gene is expressed, in some but not allcells of the animal or plant.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields, et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and International PatentPublication No. WO 98/44350.

The term “antibody” as used herein includes antibodies obtained fromboth polyclonal and monoclonal preparations, as well as, the following:hybrid (chimeric) antibody molecules (see, for example, Winter, et al.(1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)2 andF(ab) fragments; Fv molecules (non-covalent heterodimers, see, forexample, Inbar, et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; andEhrlich, et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules(sFv) (see, for example, Huston, et al. (1988) Proc Natl Acad Sci USA85:5879-5883); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack, et al. (1992) Biochem 31:1579-1584; Cumber,et al. (1992) J Immunology 149B:120-126); humanized antibody molecules(see, for example, Riechmann, et al. (1988) Nature 332:323-327;Verhoeyan, et al. (1988) Science 239:1534-1536; and U.K. PatentPublication No. GB 2,276,169, published 21 Sep. 1994); and, anyfunctional fragments obtained from such molecules, wherein suchfragments retain immunological binding properties of the parent antibodymolecule.

As used herein, the term “monoclonal antibody” refers to an antibodycomposition having a homogeneous antibody population. The term is notlimited regarding the species or source of the antibody, nor is itintended to be limited by the manner in which it is made. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)2, Fv, and other fragments, as well as chimeric and humanizedhomogeneous antibody populations that exhibit immunological bindingproperties of the parent monoclonal antibody molecule.

Nucleases

Described herein are artificial nucleases that can be used to makesingle-stranded breaks (SSBs, also referred to as “nicks”) indouble-stranded DNA. Also described herein are methods of facilitatinghomologous recombination (e.g., targeted integration) by introducing aSSB into the genome.

A. Cleavage Domains

The nucleases described herein comprise a nuclease (cleavage domain,cleavage half-domain). The cleavage domain portion of the fusionproteins disclosed herein can be obtained from any endonuclease orexonuclease. Exemplary endonucleases from which a cleavage domain can bederived include, but are not limited to, restriction endonucleases andhoming endonucleases. See, for example, 2002-2003 Catalogue, New EnglandBiolabs, Beverly, Mass.; and Belfort, et al. (1997) Nucleic Acids Res.25:3379-3388. Additional enzymes which cleave DNA are known (e.g., 51Nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease;yeast HO endonuclease; see also Linn, et al. (eds.) Nucleases, ColdSpring Harbor Laboratory Press, 1993). One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains and cleavage half-domains. For example, the cleavage domain of ameganuclease such as SceI can be rendered partially inactive to induceSSBs rather than DSBs.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof).

In addition, the target sites for the two fusion proteins are preferablydisposed, with respect to each other, such that binding of the twofusion proteins to their respective target sites places the cleavagehalf-domains in a spatial orientation to each other that allows thecleavage half-domains to form a functional cleavage domain, e.g., bydimerizing. Thus, in certain embodiments, the near edges of the targetsites are separated by 5-8 nucleotides or by 15-18 nucleotides. Howeverany integral number of nucleotides or nucleotide pairs can intervenebetween two target sites (e.g., from 2 to 50 nucleotide pairs or more).In general, the site of cleavage lies between the target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150; and 5,487,994; as wellas Li, et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li, et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Nat'l. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite, et al. (1998) Proc. Natl. Acad. Sci. USA95:10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-FokI fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-FokI fusionsare provided elsewhere in this disclosure.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPatent Publication No. WO 07/014275, incorporated herein in itsentirety. Additional restriction enzymes also contain separable bindingand cleavage domains, and these are contemplated by the presentdisclosure. See, for example, Roberts, et al. (2003) Nucleic Acids Res.31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 2005/0064474; 2006/0188987; and2008/0131962, the disclosures of all of which are incorporated byreference in their entireties herein. Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of FokI are all targets for influencing dimerizationof the FokI cleavage half-domains.

Exemplary engineered cleavage half-domains of FokI that form obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFokI and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

For example, in certain embodiments, nuclease combinations are used inwhich the cleavage domains have a mutation at 490 that replaces Glu (E)with Lys (K); a mutation at 538 that replaces Iso (I) with Lys (K); amutation at 486 that replaces Gln (Q) with Glu (E); and a mutation atposition 499 that replaces Iso (I) with Lys (K). Specifically, theengineered cleavage half-domains described herein were prepared bymutating positions 490 (E→K) and 538 (I→K) in one cleavage half-domainto produce an engineered cleavage half-domain designated “E490K:I538K”or “KK” and by mutating positions 486 (Q→E) and 499 (I→L) in anothercleavage half-domain to produce an engineered cleavage half-domaindesignated “Q486E:I499L” or “EL.”

Another example of a pair of engineered cleavage half-domains that forman obligate heterodimer (in which aberrant cleavage is minimized orabolished) is one in which one cleavage half-domain is mutated atposition 487 (R→D, also referred to as “RD”) and other cleavagehalf-domain is mutated at position 483 (D→R, also referred to as “DR”).

Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (FokI) as described in U.S. PatentPublication No. 2005/0064474 (see, e.g., Example 5); and InternationalPatent Publication No. WO 07/139898.

The cleavage half-domains of the present invention may also include onemore mutations the catalytic domain of the nuclease, which renders thecleavage half-domain inactive. Non-limiting examples of amino acids thatcan be mutated in the catalytic domain of FokI includes amino acidresidues 450, 467 and/or 469 (as determined relative to wild-type). Incertain embodiments, one or more point mutations are made in thecatalytic domain of one member of the obligate heterodimer so as toinactivate the catalytic activity of the cleavage half-domain. Forinstance, position 450 may be mutated from D to N, position 467 may bemutated from D to A; and position 469 may be mutated from K to A. Otheramino acids may be substituted at these or other positions.

B. DNA-Binding Domains

The nucleases described herein also comprise at least one DNA-bindingdomain. Any DNA-binding domain can be used in the compositions andmethods disclosed herein. In certain embodiments, the DNA binding domaincomprises a zinc finger protein. Preferably, the zinc finger protein isnon-naturally occurring in that it is engineered to bind to a targetsite of choice. See, for example, Beerli, et al. (2002) NatureBiotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal,et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000)Curr. Opin. Struct. Biol. 10:411-416. An engineered zinc finger bindingdomain can have a novel binding specificity, compared to anaturally-occurring zinc finger protein. Engineering methods include,but are not limited to, rational design and various types of selection.Rational design includes, for example, using databases comprisingtriplet (or quadruplet) nucleotide sequences and individual zinc fingeramino acid sequences, in which each triplet or quadruplet nucleotidesequence is associated with one or more amino acid sequences of zincfingers which bind the particular triplet or quadruplet sequence. See,for example, co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261,incorporated by reference herein in their entireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancementof binding specificity for zinc finger binding domains has beendescribed, for example, in co-owned International Patent Publication No.WO 02/077227.

Selection of target sites; ZFPs and methods for design and constructionof fusion proteins (and polynucleotides encoding same) are known tothose of skill in the art and described in detail in U.S. PatentPublication Nos. 2005/0064474 and 2006/0188987, incorporated byreference in their entireties herein.

In addition, as disclosed in these and other references, zinc fingerdomains and/or multi-fingered zinc finger proteins may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

Alternatively, the DNA-binding domain may be derived from a nuclease.For example, the recognition sequences of homing endonucleases andmeganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI,I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIIIare known. See also U.S. Pat. Nos. 5,420,032; 6,833,252; Belfort, et al.(1997) Nucleic Acids Res. 25:3379-3388; Dujon, et al. (1989) Gene82:115-118; Perler, et al. (1994) Nucleic Acids Res. 22, 1125-1127;Jasin (1996) Trends Genet. 12:224-228; Gimble, et al. (1996)J. Mol.Biol. 263:163-180; Argast, et al. (1998)J. Mol. Biol. 280:345-353 andthe New England Biolabs catalogue. In addition, the DNA-bindingspecificity of homing endonucleases and meganucleases can be engineeredto bind non-natural target sites. See, for example, Chevalier, et al.(2002) Molec. Cell 10:895-905; Epinat, et al. (2003) Nucleic Acids Res.31:2952-2962; Ashworth, et al. (2006) Nature 441:656-659; Paques, et al.(2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.2007/0117128.

In certain embodiments, the nucleases as described herein comprise oneDNA-binding domain and one cleavage domain, for example a zinc fingerprotein and a FokI cleavage half-domain. In other embodiments, thenuclease comprises one DNA-binding domain and two or more cleavagedomains, for example a zinc finger protein comprising one catalyticallyactive FokI cleavage half-domain and one catalytically inactivate FokIcleavage half-domain. The cleavage half-domains are able to dimerizewhen the DNA-binding domain is bound to its target site and produce asingle-stranded cut near the target site.

Delivery

The nucleases (e.g., ZFNs) described herein may be delivered to a targetcell by any suitable means. Suitable cells include but not limited toeukaryotic (animal and/or plant) and prokaryotic cells and/or celllines. Non-limiting examples of such cells or cell lines generated fromsuch cells include COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44, CHO-DUXB11,CHO-DUKX, CHOK1SV), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), and perC6cells as well as insect cells such as Spodoptera fugiperda (Sf), orfungal cells such as Saccharomyces, Pichia and Schizosaccharomyces. Incertain embodiments, the cell line is a CHO-K1, MDCK or HEK293 cellline. In addition, primary cells which may be sensitive to double strandbreaks may be used. These may include, but are not limited to, CD34+human stem cells, embryonic stem cells, mouse embryonic stem cells, andinduced pluripotent cells. Cells known to be used in the creation oftransgenic organisms, such as mouse embryonic stem cells and oocytes mayalso be used.

Methods of delivering nucleases comprising zinc finger proteins asdescribed herein are described, for example, in U.S. Pat. Nos.6,453,242; 6,503,717; 6,534,261; 6,599,692; 6,607,882; 6,689,558;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, thedisclosures of all of which are incorporated by reference herein intheir entireties.

Nucleases as described herein may also be delivered using vectorscontaining sequences encoding one or more of the nuclease(s) (e.g.,ZFNs). Any vector systems may be used including, but not limited to,plasmid vectors, retroviral vectors, lentiviral vectors, adenovirusvectors, poxvirus vectors; herpesvirus vectors and adeno-associatedvirus vectors, etc. See, also, U.S. Pat. Nos. 6,534,261; 6,607,882;6,824,978; 6,933,113; 6,979,539; 7,013,219; and 7,163,824, incorporatedby reference herein in their entireties, also described in detailconstitutive or inducible promoters that can be operably linked to thesequences encoding the nuclease(s) to drive expression of thesenucleases. Furthermore, it will be apparent that any of these vectorsmay comprise one or more nuclease encoding sequences. Thus, when one ormore pairs of ZFNs are introduced into the cell, the ZFNs may be carriedon the same vector or on different vectors. When multiple vectors areused, each vector may comprise a sequence encoding one or multiple ZFNs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs in cells (e.g.,mammalian cells) and target tissues. Such methods can also be used toadminister nucleic acids encoding ZFPs to cells in vitro. In certainembodiments, nucleic acids encoding ZFPs are administered for in vivo orex vivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson (1992) Science 256:808-813; Nabel & Felgner(1993) TIBTECH 11:211-217; Mitani & Caskey (1993) TIBTECH 11:162-166;Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; VanBrunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) RestorativeNeurology and Neuroscience 8:35-36; Kremer & Perricaudet (1995) BritishMedical Bulletin 51(1):31-44; Haddada, et al. in Current Topics inMicrobiology and Immunology Doerfler and Bohm (eds.) (1995); and Yu, etal. (1994) Gene Therapy 1:13-26.

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example U.S. Pat. No. 6,008,336).Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, International PatentPublication Nos. WO 91/17424 and WO 91/16024. Delivery can be to cells(ex vivo administration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese, etal. (1995) Cancer Gene Ther. 2:291-297; Behr, et al. (1994) BioconjugateChem. 5:382-389; Remy, et al. (1994) Bioconjugate Chem. 5:647-654; Gao,et al. (1995) Gene Therapy 2:710-722; Ahmad, et al. (1992) Cancer Res.52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975;4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher, et al. (1992) J. Virol.66:2731-2739; Johann, et al. (1992) J. Virol. 66:1635-1640; Sommerfelt,et al. (1990) Virol. 176:58-59; Wilson, et al. (1989) J. Virol.63:2374-2378; Miller, et al. (1991) J. Virol. 65:2220-2224;International Patent Publication No. WO 94/26877).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West, et al. (1987) Virology160:38-47; U.S. Pat. No. 4,797,368; International Patent Publication No.WO 93/24641; Kotin (1994) Human Gene Therapy 5:793-801; Muzyczka (1994)J. Clin. Invest. 94:1351. Construction of recombinant AAV vectors aredescribed in a number of publications, including U.S. Pat. No.5,173,414; Tratschin, et al. (1985)Mol. Cell. Biol. 5:3251-3260;Tratschin, et al. (1984)Mol. Cell. Biol. 4:2072-2081; Hermonat &Muzyczka (1984) PNAS 81:6466-6470; and Samulski et al. (1989) J. Virol.63:03822-3828.

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar, et al. (1995) Blood 85:3048-305; Kohn, etal. (1995) Nat. Med. 1:1017-102; Malech, et al. (1997) PNAS 94:2212133-12138). PA317/pLASN was the first therapeutic vector used in agene therapy trial. (Blaese, et al. (1995) Science 270:475-480).Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. (Ellem, et al. (1997) Immunol Immunother 44(1):10-20;Dranoff, et al. (1997) Hum. Gene Ther. 1:111-112.

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner, et al. (1998) Lancet 351:1702-3 (1998), Kearns. et al. (1996)Gene Ther. 9:748-55).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman, et al. (1998) Hum.Gene Ther. 7:1083-1089). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker, et al.(1996) Infection 24(1):5-10; Sterman, et al. (1998) Hum. Gene Ther.9(7):1083-1089; Welsh, et al. (1995) Hum. Gene Ther. 2:205-218; Alvarez,et al. (1997) Hum. Gene Ther. 5:597-613; Topf, et al. (1998) Gene Ther.5:507-513; Sterman, et al. (1998) Hum. Gene Ther. 7:1083-1089.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and w2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han, et al. (1995) Proc. Natl.Acad. Sci. USA 92:9747-9751, reported that Moloney murine leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney, et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba, et al. (1992) J.Exp. Med. 176:1693-1702).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+(T cells), CD45+(panB cells), GR-1 (granulocytes),and Tad (differentiated antigen presenting cells) (see Inaba, et al.(1992) J. Exp. Med. 176:1693-1702).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638. Vectors useful forintroduction of transgenes into hematopoietic stem cells, e.g., CD34⁺cells, include adenovirus Type 35.

Vectors suitable for introduction of transgenes into immune cells (e.g.,T-cells) include non-integrating lentivirus vectors. See, for example,Ory, et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull, etal. (1998) J. Virol. 72:8463-8471; Zuffery, et al. (1998) J. Virol.72:9873-9880; Follenzi, et al. (2000) Nature Genetics 25:217-222.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

As noted above, the disclosed methods and compositions can be used inany type of cell including, but not limited to, prokaryotic cells,fungal cells, Archaeal cells, plant cells, insect cells, animal cells,vertebrate cells, mammalian cells and human cells. Suitable cell linesfor protein expression are known to those of skill in the art andinclude, but are not limited to COS, CHO (e.g., CHO-S, CHO-K1, CHO-DG44,CHO-DUXB11), VERO, MDCK, WI38, V79, B14AF28-G3, BHK, HaK, NSO,SP2/0-Ag14, HeLa, HEK293 (e.g., HEK293-F, HEK293-H, HEK293-T), perC6,insect cells such as Spodoptera fugiperda (Sf), and fungal cells such asSaccharomyces, Pischia and Schizosaccharomyces. Progeny, variants andderivatives of these cell lines can also be used. In addition, primarycells which may be sensitive to double strand breaks may be used. Thesemay include, but are not limited to, CD34+ human stem cells, embryonicstem cells, mouse embryonic stem cells, and induced pluripotent cells.Cells known to be used in the creation of transgenic organisms, such asmouse embryonic stem cells and oocytes may also be used.

Applications

The disclosed compositions and methods can be used for any applicationin which introduction of a single-stranded break at a selected locationis desired. Moreover, as demonstrated herein, single-stranded cleavagefacilitates targeted integration into the site of the single-strandedbreak (via homologous recombination), without inducing non-homologousend-joining (NHEJ) events.

Thus, the compositions and methods described herein can be used for anynuclease application in which specifically targeted single-strandedcleavage is desirable and/or to replace any genomic sequence with anexogenous sequence (e.g., homologous, non-identical sequence).

For targeted integration, one or more zinc finger binding domains areengineered to bind a target site at or near the predetermined cleavagesite, and a fusion protein comprising the engineered zinc finger bindingdomain(s) and at least first and second cleavage half-domains (that forma dimer) are expressed in a cell. The first cleavage domain iscatalytically inactivated and, upon binding of the zinc finger portionof the fusion protein to the target site and dimerization of thecleavage half-domains, a single-stranded cut is made in the DNA near thetarget site.

The presence of a single-stranded break facilitates integration ofexogenous sequences via homologous recombination. Thus, a polynucleotidecomprising at least one exogenous sequence to be inserted into thegenome will typically include one or more regions of homology withtarget gene to facilitate homologous recombination.

Any sequence of interest (exogenous sequence) can be introduced asdescribed herein. Exemplary exogenous sequences include, but are notlimited to any polypeptide coding sequence (e.g., cDNAs), promoter,enhancer and other regulatory sequences, shRNA expression cassettes,epitope tags, marker genes, cleavage enzyme recognition sites andvarious types of expression constructs. In addition, the exogenousnucleic acid sequence may produce one or more RNA molecules (e.g., smallhairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs (miRNAs),etc.). Such sequences can be readily obtained using standard molecularbiological techniques (cloning, synthesis, etc.) and/or are commerciallyavailable. For example, MISSION™ TRC shRNA libraries are commerciallyavailable from Sigma Aldrich.

Marker genes include, but are not limited to, sequences encodingproteins that mediate antibiotic resistance (e.g., ampicillinresistance, neomycin resistance, G418 resistance, puromycin resistance),sequences encoding colored or fluorescent or luminescent proteins (e.g.,green fluorescent protein, enhanced green fluorescent protein, redfluorescent protein, luciferase), cell surface antigens (e.g., ANGFR)and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence.

In a preferred embodiment, the exogenous sequence comprises apolynucleotide encoding any polypeptide of which expression in the cellis desired, including, but not limited to antibodies, antigens, enzymes,receptors (cell surface or nuclear), hormones, lymphokines, cytokines,reporter polypeptides, growth factors, and functional fragments of anyof the above. The coding sequences may be, for example, cDNAs. Theexogenous sequence may also encode transcriptional regulatory factors.

For example, the exogenous sequence comprises a sequence encoding apolypeptide that is lacking or non-functional in the subject having agenetic disease, including but not limited to any of the followinggenetic diseases: achondroplasia, achromatopsia, acid maltasedeficiency, adenosine deaminase deficiency (OMIM No. 102700),adrenoleukodystrophy, aicardi syndrome, alpha-1 antitrypsin deficiency,alpha-thalassemia, androgen insensitivity syndrome, apert syndrome,arrhythmogenic right ventricular, dysplasia, ataxia telangictasia, barthsyndrome, beta-thalassemia, blue rubber bleb nevus syndrome, canavandisease, chronic granulomatous diseases (CGD), cri du chat syndrome,cystic fibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GM1),hemochromatosis, the hemoglobin C mutation in the 6^(th) codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteussyndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachmansyndrome, sickle cell disease (sickle cell anemia), Smith-Magenissyndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia AbsentRadius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberoussclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landaudisease, Waardenburg syndrome, Williams syndrome, Wilson's disease,Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP,OMIM No. 308240).

Additional exemplary diseases that can be treated by targetedintegration following single-stranded cleavage include acquiredimmunodeficiencies, lysosomal storage diseases (e.g., Gaucher's disease,GM1, Fabry disease and Tay-Sachs disease), mucopolysaccahidosis (e.g.Hunter's disease, Hurler's disease), hemoglobinopathies (e.g., sicklecell diseases, HbC, α-thalassemia, β-thalassemia) and hemophilias.

In certain embodiments, the exogenous sequences can comprise a markergene (described above), allowing selection of cells that have undergonetargeted integration, and a linked sequence encoding an additionalfunctionality.

Furthermore, although not required for expression, exogenous sequencesmay also be transcriptional or translational regulatory sequences, forexample, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

Nucleases as described herein can also be used for inactivation (partialor complete) of one or more genomic sequences. Inactivation can beachieved, for example, by targeted recombination of a missense ornonsense codon into the coding region, by targeted recombination of anirrelevant sequence (i.e., a “stuffer” sequence) into the gene or itsregulatory region, so as to disrupt the gene or regulatory region, or bytargeting recombination of a splice acceptor sequence into an intron tocause mis-splicing of the transcript.

ZFN-mediated inactivation (e.g., knockdown or knockout) of endogenousgenes can be used, for example, to generate cell lines deficient ingenes involved in apoptosis or protein production (e.g.,post-translational modifications such as fucosylation). ZFN-mediatedinactivation can also be used to generate transgenic organisms (e.g.,plants or transgenic animals).

Targeted cleavage of infecting or integrated viral genomes can be usedto treat viral infections in a host. Additionally, targeted cleavage ofgenes encoding receptors for viruses can be used to block expression ofsuch receptors, thereby preventing viral infection and/or viral spreadin a host organism. Targeted mutagenesis of genes encoding viralreceptors (e.g., the CCR5 and CXCR4 receptors for HIV) can be used torender the receptors unable to bind to virus, thereby preventing newinfection and blocking the spread of existing infections. See,International Patent Publication WO 2007/139982. Non-limiting examplesof viruses or viral receptors that may be targeted include herpessimplex virus (HSV), such as HSV-1 and HSV-2, varicella zoster virus(VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6 andHHV7. The hepatitis family of viruses includes hepatitis A virus (HAV),hepatitis B virus (HBV), hepatitis C virus (HCV), the delta hepatitisvirus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV). Otherviruses or their receptors may be targeted, including, but not limitedto, Picornaviridae (e.g., polioviruses, etc.); Caliciviridae;Togaviridae (e.g., rubella virus, dengue virus, etc.); Flaviviridae;Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae;Retroviradae; lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known asHTLV-III, LAV, ARV, hTLR, etc.) HIV-II); simian immunodeficiency virus(SIV), human papillomavirus (HPV), influenza virus and the tick-borneencephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed.1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,eds. 1991), for a description of these and other viruses. Receptors forHIV, for example, include CD4, CCR-5 and CXCR-4.

The methods and compositions as described herein can also be used in invitro contexts, for example, as a tool for the introduction of asite-specific nick in an isolated DNA template, for nucleic acidamplification (e.g., for cloning, library production), for nucleic aciddetection in any sample (e.g., diagnostics of the presence of geneticconditions and/or infectious agents), for genome walking, for analysisof DNA methylation analysis and the like. See, e.g., U.S. Pat. Nos.7,314,714; 7,112,423; 7,090,804; 6,884,586; and 6,395,523. For instance,nicking nucleases as described herein can be used alone or incombination with other enzymes to generate long and non-complementaryoverhangs as well as other structures.

In addition, the compositions as described herein can be used forgeneration of nicked (or gapped) double stranded DNA for standdisplacement amplification (SDA), an isothermal DNA amplificationapproach that provides a rapid (as short as 15 minutes) alternative todetection of polymerase chain reaction (PCR). See, e.g., U.S. Pat. No.5,523,204; Walker, et al. (1992) Nucleic Acids Res. 20(7):1691-1696. Theuse of SDA to date has depended on modified phosphorthioate nucleotidesin order to produce a hemi-phosphorthioate DNA duplex that on themodified strand would be resistant to enzyme cleavage, resulting inenzymatic nicking instead of digestion to drive the displacementreaction or on engineered “nickase” enzymes that do not cutdouble-stranded DNA. Accordingly, the nucleases described herein can bereadily adapted for any application in which SDA is currently applied.Thus, the compositions of the invention can be used in detection ofnucleic acid sequences and, accordingly, in the diagnosis of thepresence of genetic diseases and/or infectious agents, such as HPV,Hepatitis virus (HCV, HBV, HAV), HIV, Mycobacterium tuberculosis andChlamydia trachomatis, from any sample (e.g., blood, urine, plasma,tissue samples, isolated cells, fixed cells, etc.).

Additional in vitro applications include the generation ofexonucleolytic degradations. For example, nicking nucleases as describedherein can be used in combination with Si for the creation of nesteddeletions. See, e.g., U.S. Pat. Nos. 7,244,560; 6,867,028; and6,828,098.

EXAMPLES Example 1: Design and Construction of ZFNs with Mutant CleavageDomains

Zinc finger proteins targeted to CCR5 as described in InternationalPatent Publication No. WO 2007/139982 were operably linked to engineeredcleavage domains as described in U.S. Patent Publication No.2008/0131962, essentially as described in U.S. Patent Publication No.2005/0064474 and International Patent Publication WO 2005/014791.

Specifically, CCR5-binding zinc finger protein designated 8196z wasfused to either the obligate heterodimer forming cleavage domain having(1) E490K and I538K mutations to form the zinc finger nuclease (ZFN)designated “8196zKK” or (2) an D483R mutation to produce the ZFNdesignated “8196zDR.” Similarly, the CCR5-binding zinc finger proteindesignated 8267 was fused to either the obligate heterodimer formingcleavage domain having (1) Q486E and I499L mutations to form the ZFNdesignated “8267EL” or (2) an R487D mutation to form the ZFN designated“8267RD.” Catalytically inactive forms of all four ZFNs were alsoprepared by site-directed mutagenesis of amino acid residues 450 (D toN, designated D450N); 467 (D to A, designated D467A); or 469 (K to A,designated K469A).

Example 2: Dimers of Cleavage Domains with One Catalytically InactiveCleavage Domain Induce Single-Stranded Breaks

The ZFNs described in Example 1 were used in various pairwisecombinations and cleavage events evaluated. In particular, ZFN variantsdescribed in Example 1 were synthesized using TNT T7 Quick™ coupledtranscription/translation system (Promega).

The appropriate substrate containing the ZFNs target sites was generatedby PCR amplification of CCR5 sequences flanking CCR5 ZFN binding regionto generate a 292 base pair substrate. The substrates were ³²Pend-labeled using T4 polynucleotide kinase and incubated with thefollowing ZFN pairs: 8196zKK and 8267EL (WT); 8196zKK and 8267EL, where8267 EL includes catalytically inactivating point mutation D450N(D450N); 8196zKK and 8267EL, where 8267 EL includes catalyticallyinactivating point mutation D467A (D467A); 8267RD and 8196zDR (WT);8267RD and 8196zDR, where 8196zDR includes catalytically inactivatingpoint mutation D450N (D450N); and 8267RD and 8196zDR, where 8196zDRincludes catalytically inactivating point mutation D467A (D467A). Themixture of radio-labeled substrate DNA and ZFN proteins was incubated at37° C. for 2 hr as described previously (Miller, et al. (2007) Nat.Biotech. 25:778-785) with modifications described below.

Cleaved DNA were extracted by phenol/chloroform and either untreated(double-stranded cleavage products) or treated with a DNA denaturingsolution (1.0M glyoxal, 10 mM NaH₂PO₄/Na₂HPO₄, pH 7.0, 50% DMSO) togenerate single-stranded DNA before separation on a 10% Ready™ gel TBEgel (Invitrogen).

As shown in FIG. 2A, double-stranded cleavage products were efficientlygenerated only with ZFN pairs 8196zKK+8267EL (WT) or 8267RD+8196zDR(WT), in which the FokI cleavage half-domains of both left and rightZFNs are catalytically active. The parental uncut DNA (292 bp) ispresent with all ZFN pairs. Two fragments (˜168 bp and ˜124 bp) wereseen with when both ZFNs were catalytically active (8196zKK+8267EL (WT)or 8267RD+8196zDR (WT) pairs). For all ZFN pair combination in which oneof the ZFNs is catalytically inactivated by the indicated pointmutation, double-stranded breaks in the CCR5 target DNA were notgenerated.

However, as shown in FIG. 2B, ZFN pairs with one catalytically inactiveZFN induced single-stranded breaks. In particular, the ˜168 bp fragmentseen when both FokI cleavage half-domains are catalytically active indouble-stranded cleavage products (FIG. 2A, lanes 3 and 8 from left) wasalso seen in single-stranded cleavage products treated with ZFN pairs8196zKK+8267EL D450N and 8196zKK+8267EL D467A (containing onecatalytically inactive cleavage domain). See, FIG. 2B, lanes 4 and 5from left. The smaller ˜124 bp fragment is not visible because only the5′ end of each DNA strand was end-labeled (see, FIG. 2C).

Similarly, the ˜124 bp fragment seen when both FokI cleavagehalf-domains are catalytically active in double-stranded cleavageproducts (FIG. 2A, lanes 3 and 8 from left) was also seen insingle-stranded cleavage products treated with 8267RD+8196zDR D450N or8267RD+8196zDR D467A (each containing a catalytically inactive cleavagehalf-domain). See, FIG. 2B, lanes 9 and 10 from left. Again, the larger˜168 bp fragment is not visible in these samples because only the 5′ endof each DNA strand was end-labeled (see, FIG. 2C).

These results demonstrate that the use of dimers of cleavagehalf-domains in which one cleavage half-domain is catalyticallyinactivated generates SSBs/nicks in double-stranded DNA.

Example 3: Repairing SSB/Nicks by Single-Strand Annealing in Cells A.Yeast Cells

To evaluate whether SSB/nicks can be used as an alternative method forinducing recombination-based genome editing at a targeted locus, wefirst tested the system in yeast, essentially as described in Doyon, etal. (2008) Nat Biotechnol 26:702-8 and U.S. Patent Publication No.2009/0111119.

A SSA-MEL1 reporter yeast strain, in which the CCR5 ZFN-targeting siteswas introduced between the 2 overlapping and nonfunctional fragments ofMEL1 gene, was transformed with the ZFN expression plasmids:ZFN-L-EL-D450N+ZFN-R-KK (D450N), ZFN-L-EL-D467A+ZFN-R-KK (D467A) orcontrol vectors (Control). ZFN expression was induced by culture cellsin 2% galactose for 2 to 6 hours before overnight culturing in glucosemedia and assaying for galactosidase activity.

Significantly increased galactosidase activities were observed alongwith the induction of ZFN expression for 2-6 hours by galactose incultures treated with D450N/WT (40.0-75.5 mU) or D467A/WT (51.7-96.0 mU)ZFNs compared to cultures treated with the control vectors (2.7-4.1 mU),demonstrating that SSB/nicks induced by ZFNs as described herein arerecombinogenic and can be repaired by single-strand annealing in yeast.

To ensure that ZFNs induced MEL1 expression via SSA repair of SSB/nicksand not DSBs, the ability of the ZFNs to induce DSB formation was alsoevaluated. In the absence of homologous template sequence, DSB inductionis lethal to more than 99.8% of the yeast cells in a colony. Yeast cellswith the CCR5 ZFN target sites integrated into the HO locus weretransformed with ZFN expression plasmids and cultured in minimal mediacontaining glucose or galactose at ten-fold serial dilutions. Only cellsin which the ZFNs do not induce DSBs would be expected to surviveinduction of ZFN expression.

The cells grew well in the presence of glucose (no induction of ZFNexpression) regardless of the ZFN expression plasmid introduced in thesecells. In the presence of galactose, we observed a 2-3 log reduction inthe number of yeast cells that survived in culture when treated with theWT/WT ZFNs, as compared to cultures treated with control vectors. Incontrast, yeast transformed with an expression construct replacing oneof the WT/WT ZFNs with one of the ZFNs variants (D450N or D467A) wereindistinguishable from those cultures treated with the control vectors.Western blot analysis of extracts taken from these cultures confirmedthat the expression levels of the ZFN proteins were similar.

As the observed lethality is completely dependent on the presence of theintegrated CCR5 target sites, it is not due to the generation of randomDSBs by the wild-type ZFN. Therefore, these results confirmed thatwithin the context of a living cell, the ZFNs generating SSB induceSSA-mediated repair via SSB/nick formation and do not catalyze DSBs.

B. Mammalian Cells

To evaluate whether SSB/nicks can be repaired by an HDR-dependent SSApathway in mammalian cells, K562 cells were co-transfected with ZFNexpression plasmids and a SSA-GFP reporter plasmid, in which the eGFPopen reading frames with repeated homologous sequences flank a stretchof DNA encoding the CCR5 ZFN target sites (required for HDR dependency),and the number of cells undergoing SSA-mediated repair and expressingGFP was then monitored by flow cytometric analysis.

As shown in FIG. 3, significantly more GFP⁺ cells were observed insamples treated with D450N/WT (ZFN-L-EL-D450N+ZFN-R-KK+SSA-GFP reporter,3.55%) or D467A/WT (ZFN-L-EL-D467A+ZFN-R-KK+CCR5-SSA-GFP reporter,3.40%) than the sample treated with the SSA-GFP reporter alone (0.67%),though it is 2-3 fold less compared to the sample treated with WT/WTZFNs (ZFN-L-EL+ZFN-R-KK+SSA-GFP reporter, 8.58%).

These data indicate that SSB/nicks generated by ZFNs generating SSBs canalso be repaired by the SSA pathway in mammalian cells.

Example 4: Single-Stranded Breaks Facilitate Target Integration byHomologous Recombination

The ability of ZFNs that induce SSB/nicks to facilitate homologousrecombination and/or NHEJ was also determined.

Briefly, K562 cells were treated with ZFN combinations indicated inTable 1 alone or with ZFN combinations indicated in Table 1 and a CCR5patch donor sequence. Genomic DNAs were collected from treated cellsafter 3 days and then tested for NHEJ events by the Surveyor™ nucleaseassay and TaqMan™ qPCR assay. In addition, an RFLP assay was used totest targeted integration by homologous recombination.

A. Non-Homologous End Joining

As noted above, the percentage of NHEJ events was estimated by Surveyor™nuclease assay and TaqMan™ qPCR assay. Briefly, the Surveyor™ nucleaseassay was conducted as described in Miller, et al. (2007) Nat. Biotech.25:778-785). The Taqman™ qPCR assay was conducted according to themanufacturer's instruction to measure the presence of a 5 bp insertionknown to occur in approximately 10 to 30% of cells followingdouble-stranded cleavage with CCR5 ZFNs. Accordingly, specific primerswere designed based on this pentamer insertion event, tested, andoptimized. The assay is able to detect 1 copy of the plasmid DNAcontaining the pentamer insertion sequence. The assay can also easilydetect 0.01% (le-4) NHEJ event in genomic DNA samples.

In addition to the uncut parental DNA (292 bp), the presence of thecleaved large fragment (˜168 bp) and small fragment (˜124 bp) indicatescleavage by the Surveyor™ nuclease due to NHEJ-induced modification ofDNA sequences and the subsequent formation of DNA heteroduplex duringthe assay process.

Results, as shown in Table 1 and FIG. 4A, demonstrate that thecombination of wild-type (WT) ZFNs, 8196zKK+8267EL or 8267RD+8196zDR(lanes labeled 1 and 9), induced DSB, of which 54.3% and 36.7% wererepaired by NHEJ, respectively. See, also, FIG. 5B showing NHEJ onlywith wild-type ZFNs.

By contrast, none of the other ZFN combinations, in which one of the twoZFNs contains catalytically inactivated point mutations in the FokIdomain, induced double-stranded breaks, based on the absence of repairby NHEJ as detected by a radioactive Surveyor™ nuclease assay and ahighly sensitive Taqman™ qPCR assay. See, FIG. 5B.

TABLE 1 Estimated NHEJ % Estimated Nuclease Taqman Sample# Left ZFNRight ZFN TI % assay qPCR 1 8267EL wt 8196z-KK wt 29.4 54.3 55.3 28267EL wt 8196z-KK D467A 1.1 0.0 0.0 3 8267EL wt 8196z-KK K469A 0.0 0.00.0 4 8267EL D467A 8196z-KK wt 1.6 0.0 0.0 5 8267EL K469A 8196z-KK wt0.7 0.0 0.0 6 8267EL D467A 8196z-KK D467A 0.0 0.0 0.0 7 8267EL K469A8196z-KK K469A 0.0 0.0 0.0 8 8267EL D467A 8196z-KK K469A 0.0 0.0 0.0 98267RD wt 8196z-DR wt 28.4 36.7 34.6 10 8267RD wt 8196z-DR D467A 0.7 0.00.0 11 8267RD wt 8196z-DR K469A 0.0 0.0 0.0 12 8267RD D467A 8196z-DR wt2.0 0.0 0.0 13 8267RD K469A 8196z-DR wt 0.5 0.0 0.0 14 8267RD D467A8196z-DR D467A 0.0 0.0 0.0 15 8267RD K469A 8196z-DR K469A 0.0 0.0 0.0 168267RD K469A 8196z-DR D467A 0.0 0.0 0.0 17 8267EL wt 8196z-KK D467A-K4690.6 0.0 0.0 18 8267EL wt 8196z-KK D450A 1.9 0.0 0.0 19 8267EL D450A8196z-KK wt 2.5 0.0 0.0 20 8267EL D467A-K469 8196z-KK wt 0.0 0.0 0.0 218267RD wt 8196z-DR D450A 1.1 0.0 0.0 22 8267RD wt 8196z-DR D450N 1.2 0.00.0 23 8267RD D450N 8196z-DR wt 1.5 0.0 0.0 24 8267RD D467A-K4698196z-DR wt 0.9 0.0 0.0

Thus, DNA SSBs/nicks induced by ZFN pairs in which one ZFN iscatalytically inactivated as described herein are unable to induce NHEJ.

B. Targeted Integration

Targeted integration of various donors was also evaluated. Donors testedincluded a smaller 46 pb CCR5-patch donor (R5-patch donor), which has aBglI restriction enzyme site and total 46 bp insertion between the 2CCR5 ZFN binding sites; a 1.6 kb CCR5-GFP donor, in which an eGFPexpression cassette is flanked by sequence homologous to CCR5 (R5-GFPdonor); and an additional donor that replaced the eGFP marker betweenthe homologous CCR5 sequence of the CCR5-GFP donor with a truncated NGFRreporter gene and placed the eGFP marker cassette outside the homologousCCR5 sequence on the donor template plasmid (CCR5-NGFR-outGFP donor).Unless specifically indicated below, the following ZFNs were used:8267-EL (or ZFN-L-EL), 8267-EL-D450N (or ZFN-L-EL-D450N), 8267-EL-D467A(ZFN-L-EL-D467A), and 8196zKK (ZFN-R-KK).

In the presence of the smaller 46 bp CCR5-patch donor, targetedintegration was evaluated by a restriction fragment length polymorphism(RFLP) assay, in which the BglI digested wild-type PCR product is 2433bp, and the BglI digested fragments of patch-containing modified CCR5PCR products are 1554 bp and 925 bp, respectively.

As shown in FIG. 4B and Table 1, the combination of wild-type (WT) ZFNs,8196zKK+8267EL or 8267RD+8196zDR induced DSB, of which 29.4% and 28.4%were repaired by homologous recombination, respectively. The majority ofthe combinations that contained one wild-type ZFN with a catalyticallyactive FokI domain induced homologous recombination in 0.5-2.5% of theDNA repair events, whereas none of the combinations that contain 2 ZFNswith catalytically inactivated FokI domains were able to inducehomologous recombination. Furthermore, all single ZFNs, either havingwild-type catalytic activity or catalytically inactivated mutants,failed to induce homologous recombination by themselves (FIGS. 4C and4D).

In addition, as shown in FIG. 5A in a separate experiment, K562 cellstreated with the WT/WT ZFNs (DSB-inducing) showed targeted integration(TI) of the CCR5-patch donor in 30.6% of the endogenous CCR5 alleles.The cells treated with the D450N/WT or D467A/WT ZFNs also showed TI atthe CCR5 locus (7.3 and 8.0%, respectively), but at a ˜4-fold reducedlevel of efficiency. TI could not be detected in any of the samplestreated with a single ZFN (WT, D450N, or D467A).

Genotyping of single cell clones derived from unsorted pools in theexperiment using the CCR5-patch donor was also performed. K562 cellswere co-transfected with the donor DNA indicated in Table 2 andcombinations of ZFN expression plasmids: ZFN-L-EL+ZFN-R-KK (WT),ZFN-L-EL-D450N+ZFN-R-KK (D450N), or ZFN-L-EL-D467A+ZFN-R-KK (D467A).Unsorted or sorted pools were subjected to single cell cloning bylimited dilution. Single cell-derived clones were selected undermicroscope and tested by subsequent genotyping analysis.

As shown in Table 2, more than 25% of the expanded clones wereheterozygous for targeted integration into the CCR5 locus, confirmingthe high frequency of HDR-driven genome modification via induction ofSSB/nicks in K562 cells.

Similarly, the larger 1.6 kb CCR5-GFP donor described above wasintroduced into K562 cells along with the WT/WT CCR5 ZFN and nickasemutant ZFN pairs to evaluate targeted integration of the GFP expressioncassette into the CCR5 locus. Briefly, GFP⁺ cells were sorted for byfluorescence activated cell sorting (FACS) at facilities in Universityof California (Berkeley, Calif.) as described in Moehle, et al. (2007)Proc Natl Acad Sci USA 104:3055-3060, and this sorted population wasused to generate single cell-derived clones.

Among the 75 GFP+ clones derived from the sample treated with the WT/WTZFN (ZFN-L-EL+ZFN-R-KK+CCR5-GFP), 70 clones (93.3%) harbored TI eventsand 58 clones (77.3%) harbored NHEJ events. In contrast, the clonesmodified by treatment with the D450N/WT ZFNs(ZFN-L-EL-D450N+ZFN-R-KK+CCR5-GFP) showed no NHEJ-based modification,whereas 61 (65.6%) of the total 93 clones exhibited a TI event. One outof 95 clones (1.1%) derived from the cells treated with D467A/WT ZFNs(ZFN-L-EL-D467A+ZFN-R-KK+CCR5-GFP) showed an NHEJ-like mutation at CCR5,whereas 67 clones (70.5%) exhibited a TI event. See, Table 2.

For experiments using the CCR5-tNGFR-outGFP donor, cells undergoingHDR-driven TI of the sequence encoded on the donor should only expressthe NGFR marker, but not GFP (NGFR+GFP−). Expression of eGFP (GFP+)would indicate the presence of episomal DNA or random integration of thedonor plasmid at unknown sites.

To enrich cells with surface NGFR expression, cells were first incubatedwith anti-NGFR mAb (BD Pharmingen), washed, and then incubated with goatanti-mouse IgG conjugated with Dynal beads provided in the CELLectionPan mouse IgG kit (Invitrogen), followed by passing through magneticfields. Beads were then removed from enriched cells by DNase I digestionfollowing manufacture's instruction (Invitrogen). Alternatively, cellswere either unstained or incubated with anti-NGFR mAb conjugated with PE(BD Pharmingen), then sorted based on GFP and/or NGFR expression levelsusing a flow cytometric cell sorter.

The sorted NGFR+GFP−cell pool derived from cells treated with WT/WT ZFNs(ZFN-L-EL+ZFN-R-KK+CCR5-tNGFR-outGFP) contained a high level (63.2%) ofNHEJ-based modification, whereas no NHEJ modification was detected incells treated with the following ZFNs: D450N/WT(ZFN-L-EL-D450N+ZFN-R-KK+CCR5-tNGFR-outGFP) or D467A/WT(ZFN-L-EL-D467A+ZFN-R-KK+CCR5-tNGFR-outGFP), as determined by theSurveyor™ nuclease assay (FIG. 6A). This difference in the level ofmodification by NHEJ was also observed both in the sorted NGFR+GFP+ cellpopulation and unsorted pools.

While the cells treated with the CCR5-tNGFR-outGFP donor andnick-inducing ZFNs (D450N/WT or D467A/WT) did not exhibit NHEJ using theSurveyor™ nuclease assay, we found >18% of the CCR5 alleles in thesorted NGFR+GFP− cells derived from these samples harbored a targetedinsertion event, as detected by Southern blot (FIG. 6B). Furthermore,single cell-derived clones generated from samples treated with theD450N/WT and D467A/WT ZFNs confirmed that 71 out of 91 (78%) and 74 outof 96 (77.1%) clones have TI in their genomes, respectively (Table 2).In contrast, no TI events were seen in the sorted NGFR+GFP+ cells, whichwould enrich for cells that had undergone random integration of thedonor plasmid, derived from the ZFN-treated samples. Multiple Southernblot bands were seen in the sorted NGFR+GFP+ cells derived from theWT/WT ZFN (ZFN-L-EL+ZFN-R-KK+CCR5-tNFR-outGFP)-treated sample, inaddition to the expected TI band, presumably caused by the presence ofvarious kinds of NHEJ modification at CCR5, random integration of thedonor DNA template, or stable persistence of the donor plasmid asepisomal DNA.

As shown in Table 2, more than 25% of the expanded clones wereheterozygous for targeted integration into the CCR5 locus, confirmingthe high frequency of HDR-driven genome modification via induction ofSSB/nicks.

TABLE 2 Total TI TI NHEJ NHEJ Pools Donor ZFN clones clones clones %clones clones % Unsorted R5-Patch D450N 283 73 25.8% ND ND UnsortedR5-Patch D467A 284 86 30.3% ND ND GFP⁺ R5-GFP WT 75 70 93.3% 58  77.3% GFP⁺ R5-GFP D450N 93 61 65.6% 0 0.0% GFP⁺ R5-GFP D467A 95 67 70.5% 11.1% NGFR⁺GFP⁻ R5-NGFR-outGFP WT 83 79 95.2% ND ND NGFR⁺GFP⁻R5-NGFR-outGFP D450N 91 71 78.0% ND ND NGFR⁺GFP⁻ R5-NGFR-outGFP D467A 9674 77.1% ND ND

Thus, single-stranded breaks (nicks) in DNA can induce homologousrecombination in mammalian (e.g., human) cells, and can be used fortargeted integration of any DNA sequences, with reduction or eliminationof on or off-target NHEJ mutations at the ZFN cleavage sites.

Example 5: Solexa® Deep Sequencing

To further evaluate whether SSB/nicks are repaired by the NHEJ pathway,we performed Solexa® deep sequencing of the CCR5 target locus fromWT/WT, D450/WT ZFNs, or control treated K562 cells. Briefly, genomicDNAs were amplified use a pair of CCR5 primers located outside the CCR5homologous region of donor molecules. The amplified 2.5 kb CCR5fragments were gel purified and used as templates for inside PCRreactions using primers containing BpmI and XhoI restriction enzymesites.

The amplicons were then digested with BpmI and XhoI to remove the 5′-endof 16 bp of the PCR products to allow sequencing to begin close to theputative ZFN cleavage sites. The digested products were gel purified andligated to adaptors which have BpmI- or XhoI-digested DNA-like ends andcontaining no tag or a three-nucleotide ‘bar-code’ unique to eachexperiment. Adaptor-ligated PCR products were then gel purified and PCRamplified using Illumina Genomic DNA Primers (Illumina). The resultingPCR products were subjected to Solexa® deep sequencing located atCalifornia Institute for Quantitative Biosciences, University ofCalifornia (Berkeley, Calif.). A custom-written computer script was usedto extract all sequences which were either WT or consistent with NHEJmediated deletions or insertions.

Greater than 485,000 quality sequence reads (>99% confidence for eachbase) were analyzed from each of the samples tested. The sequencesderived from the WT/WT ZFN sample (ZFN-L-EL+ZFN-R-KK+CCR5-patch) showedthat 260,509 out of 713,186 sequences (36.5%) appeared to be modified byNHEJ. See, Table 3.

TABLE 3 Solexa ® deep sequencing of unsorted pools Uni- Uni- ZFN-L ZFN-RTotal WT Ins Ins Del Del Ins + Del Ins + Del % none none 1772351 17722560 0 24 14 24 0.0014 EL KK 713186 427922 68594 91 191915 500 26050936.5275 EL-D450N KK 944605 944492 18 2 25 13 43 0.0046 EL none 549766549698 8 2 24 11 32 0.0058 EL-D450N none 495811 495766 0 0 0 0 0 0.0000none KK 485162 485103 6 1 10 7 16 0.0033 * “WT” refers to wild-type;“Ins” refers to insertions; “Del” refers to deletions; “Uni” refers tounique insertions or deletions

In sharp contrast, the sequences analyzed from the D450/WT ZFNs(ZFN-L-EL-D450N+ZFN-R-KK+CCR5-patch) revealed that only 43 out of944,605 sequences (0.0046%) exhibited mutations consistent with NHEJ,a >7,900 fold reduction in NHEJ. The mutation rate in the D450/WT ZFNtreated cells is within the background noise of the assay (0-0.0058%),which is presumably caused by errors generated during the PCRamplification and sequencing steps, based on data from samples treatedwith donor DNA only or donor and a single ZFN. This is also supported bythe small number of unique sequence variants identified in the controland ZFN samples. A greater diversity in the types of sequencemodifications observed would be expected, as seen in the WT/WTZFN-treated sample, if these modifications were due to NHEJ.

The cells treated with D450N/WT ZFNs in the presence of CCR5-patch donorharbored 7.3% targeted integration events, meaning there is a >1,500fold preference for HDR over NHEJ using the ZFNs. It should be notedthat the addition of a single ZFN does not cause modification of thelocus by NHEJ, providing additional evidence that the binding of 2 ZFNsin the appropriate orientation on DNA is required for DSB formation.

These data suggested that there are either no or extremely low levels ofNHEJ events in SSB-inducing ZFN-treated samples. These data furtherconfirm that SSB/nicks are selectively repaired by HDR and not NHEJ.

Example 6: Genome-Wide Assessment of DSB Formation Induced by ZFN Pairswith a Catalytically Inactivated ZFN

SSB/nicks are one of the most frequent types of DNA damage produced byendogenous reactive oxygen species or during DNA metabolism, such as DNArepair and replication, which can be repaired precisely by DNApolymerases and ligases using the intact opposite strand as a template.See, Caldecott (2008) Nat Rev Genet 9:619-31. To further evaluate thepotential for SSB-generating ZFNs to induce a low level of DSBs atgenomic sites other than the intended target site, we performed agenome-wide assessment of DSB formation by detecting γH₂AX and 53BP1expression, since γH₂AX and 53BP1 are recruited to the site of DSBs asthe natural response to DNA damage.

Briefly, for intracellular staining of γH2AX, cells collected at varioustime points (e.g., 1, 2, 3 and 7 days) post-nucleofection werepermeabilized with perm/wash buffer (0.05% Saponin, 2.5% FBS, and 0.02%NaN3 in PBS) and then incubated with anti-γH2AX monoclonal antibody(Upstate) followed by incubation with Alexa Fluor488-conjugated goatanti-mouse immunoglobulin (Ig, Invitrogen). Cells were then analyzedusing a Guava Easycyte® single cell analysis system (Guava®Technologies).

For 53BP1 immunocytochemistry, cells were collected to prepare slides bycytospin (Thermo Scientific) and stained with anti-53BP1 rabbitpolyclonal antibodies (Bethyl Laboratories) followed by taking pictureswith a CCD camera connected to an immunofluorescence microscope (Nikon)as described before in detail in Perez, et al. (2008) Nat Biotechnol26:808-16.

Under the experimental conditions used to transfect cells with ZFNs anddonor, a significant amount of γH₂AX expression was observed in cellstreated with WT/WT ZFNs (ZFN-L-EL+ZFN-R-KK+CCR5-patch, 14.70% γH₂AX⁺) at2 days post-transfection, but not in cells treated with D450N/WT(ZFN-L-EL-D450N+ZFN-R-KK+CCR5-patch, 0.33% γH₂AX⁺). However, slightlyhigher γH₂AX expression was observed in cells treated with D467A/WT(ZFN-L-EL-D467A+ZFN-R-KK+CCR5-patch, 4.34% γH₂AX⁺), consistent with theprevious observation that the D467A/WT pair may retain a small amount ofDSB activity. As expected, more 53BP1⁺ foci were also observed in cellstreated with WT/WT ZFNs (ZFN-L-EL+ZFN-R-KK+CCR5-patch, 6.09±1.07foci/cell, Ave±SD) than in cells treated with the D450N/WT pair(ZFN-L-EL-D450N+ZFN-R-KK+CCR5-patch, 0.84±0.17 foci/cell), which isessentially at the level of background. A moderate increase in thenumber of 53BP1⁺ foci was observed in cells treated with D467A/WT(ZFN-L-EL-D467A+ZFN-R-KK+CCR5-patch, 1.77±0.33 foci/cell), compared tothe control cells transfected with the CCR5-patch donor alone (0.82±0.22foci/cell). The expression of γH₂AX and 53BP1 returned to backgroundlevels within a week after transfection (FIG. 7).

The absence of upregulated γH₂AX and 53BP1 expression under theseexperimental conditions further confirm that ZFNs as described herein donot increase the number of DSBs formed over background and providesadditional support that the targeted integration observed in theSSB-inducing ZFN-treated cells does occur through nick-induced HDR, notDSB-induced HDR, and providing an alternative method for editing thegenome of human cells while reducing the potential for both on-targetand off-target mutagenesis via NHEJ.

Example 7: SSB-ZFN-Initiated Targeted Integration in a Non-CCR5Endogenous Locus

To further evaluate whether ZFNs that induce a SSB can be used forgenome editing at loci other than the CCR5 locus, we tested D450NZFNs-initiated targeted integration at the CXCR4 locus. WT and D450NZFNs including zinc finger domains targeted to CXCR4 were prepared asdescribed in U.S. Pat. No. 8,871,905 and U.S. Patent Publication No.2015/0110757. Subsequently, K562 cells were nucleofected with aCXCR4-patch donor DNA in the absence (No ZFN) or presence of CXCR4 D450NZFNs: CXCR4-ZFN-L-EL-D450N+CXCR4-ZFN-R-KK. Cells were allowed to recoverfor 4-7 days and then nucleofected again with the same DNAs. The processwas repeated for a total of 4 nucleofections. Cells were then collected3 days after the last nucleofection for gDNA preparation and RFLP assay.

As shown in FIG. 8, D450N/WT ZFN pairs specifically targeting theendogenous CXCR4 gene located on chromosome 2 in the human genome canalso mediate a significant amount of TI at the CXCR4 locus.

These data suggests that the strategy of using targeted SSB-inducingZFNs to mediate HDR-driven genome editing may be broadly applicable andcould be universally applied across different target loci.

Example 8: Detection of Nucleic Acid Sequences

Blood or cell samples suspected of containing an infectious agent orfrom an individual suspected of having a genetic disease are collectedaccording to standard techniques. Appropriate primers are prepared for atarget nucleotide sequence that is characteristic of the genetic diseaseor infectious agent. Nucleases as described herein are constructed toinduce a site-specific single-stranded nick in the target sequenceand/or amplification primer.

The samples are incubated with the appropriate primers, nucleotides(dNTPs for amplification) and enzyme mix (containing the appropriatenicking nucleases and a DNA polymerase) such that, when present, thesequence characteristic of the genetic disease or infectious agent isamplified. One or more components may detectably labeled for ease ofdetection.

The amplified sequence, if any, is then detected using standardtechniques, for example, gel electrophoresis, flow cytometry,radiolabeling, etc. The presence of an amplified sequence is indicativeof the presence of the genetic disease or infectious agent.

Example 9: Detection of Nucleic Acid Sequences

Blood or cell samples suspected of containing an infectious agent orfrom an individual suspected of having a genetic disease are collectedaccording to standard techniques.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

1.-11. (canceled)
 12. A method of inserting an exogenous sequence into aregion of interest in a cell, the method comprising: generating asingle-stranded break in the region of interest by providing a proteincomplex including a fusion protein comprising a DNA-binding domain andat least one catalytically inactive cleavage domain or cleavagehalf-domain to the cell; and inserting the exogenous sequence into thesingle stranded break in the region of interest, thereby inserting theexogenous sequence into the region of interest in the cell.
 13. Themethod of claim 12, wherein the exogenous sequence replaces a wild-typegenomic sequence.
 14. The method of claim 12, wherein the insertion ofthe exogenous sequence partially or fully inactivates a target sequencewithin the region of interest.
 15. The method of claim 12, wherein theprotein complex further comprises a second fusion protein comprising azinc finger domain and a catalytically active cleavage half domain andthe catalytically inactive cleavage half domain forms a heterodimer withthe catalytically active cleavage half-domain of the second fusionprotein.
 16. The method of claim 15, wherein the heterodimer generatesthe single-stranded break in the region of interest.
 17. The method ofclaim 12, wherein the region of interest is a double-stranded sequence.18. The method of claim 17, wherein the double-stranded sequence iscellular chromatin.
 19. A method for replacement of a region of interestin a cell with a first nucleotide sequence comprising: (a) engineering afirst zinc finger binding domain to bind to a second sequence in theregion of interest; (b) providing a second zinc finger binding domain tobind to a third sequence in the region of interest; (c) expressing afirst fusion protein in a cell, the first fusion protein comprising thefirst zinc finger binding domain and a first catalytically activecleavage half-domain; (d) expressing a second fusion protein in thecell, the second fusion protein comprising the second zinc fingerbinding domain and a second catalytically inactive cleavage half-domain;and (e) contacting the cell with a polynucleotide comprising the firstnucleotide sequence.
 20. The method of claim 19, wherein the region ofinterest comprises cellular chromatin.
 21. The method of claim 19,wherein the first fusion protein binds to the second sequence and thesecond fusion protein binds to the third sequence, thereby positioningthe cleavage half-domains such that a single-stranded break is made incellular chromatin in the region of interest and a nucleotide sequencein the region of interest is replaced with the first nucleotidesequence.
 22. The method of claim 21, wherein the single-stranded breakin cellular chromatin is made in the region of interest at a sitebetween the second and third sequences.
 23. The method of claim 19,wherein the zinc finger nucleases are provided to the cells as proteinsand/or as one or more polynucleotides encoding said zinc fingernucleases.
 24. The method of claim 23, wherein a single polynucleotidecomprises sequences encoding both the first and the second fusionprotein.